(Nano)platforms in bladder cancer therapy: Challenges and opportunities

Abstract Urological cancers are among the most common malignancies around the world. In particular, bladder cancer severely threatens human health due to its aggressive and heterogeneous nature. Various therapeutic modalities have been considered for the treatment of bladder cancer although its prognosis remains unfavorable. It is perceived that treatment of bladder cancer depends on an interdisciplinary approach combining biology and engineering. The nanotechnological approaches have been introduced in the treatment of various cancers, especially bladder cancer. The current review aims to emphasize and highlight possible applications of nanomedicine in eradication of bladder tumor. Nanoparticles can improve efficacy of drugs in bladder cancer therapy through elevating their bioavailability. The potential of genetic tools such as siRNA and miRNA in gene expression regulation can be boosted using nanostructures by facilitating their internalization and accumulation at tumor sites and cells. Nanoparticles can provide photodynamic and photothermal therapy for ROS overgeneration and hyperthermia, respectively, in the suppression of bladder cancer. Furthermore, remodeling of tumor microenvironment and infiltration of immune cells for the purpose of immunotherapy are achieved through cargo‐loaded nanocarriers. Nanocarriers are mainly internalized in bladder tumor cells by endocytosis, and proper design of smart nanoparticles such as pH‐, redox‐, and light‐responsive nanocarriers is of importance for targeted tumor therapy. Bladder cancer biomarkers can be detected using nanoparticles for timely diagnosis of patients. Based on their accumulation at the tumor site, they can be employed for tumor imaging. The clinical translation and challenges are also covered in current review.


| INTRODUCTION
Urological cancers are responsible for high mortality around the world, and bladder cancer is the most common tumor in the urinary system. There are two major forms of bladder cancer including muscle-invasive bladder cancer (MIBC) and nonmuscle invasive bladder cancer (NMIBC). Incidence of metastasis in NMIBC is rare, albeit the chance of recurrence is 50%-70% after transurethral resection. 1 Approximately 10%-20% of cases are MIBC, and risk of metastasis is high in certain MIBC patients which is responsible for short survival in nearly up to 30% of patients. 2,3 Following a radical cystectomy, some MIBC patients demonstrate lymphatic metastasis that can lead to mortality within 5 years after diagnosis. 4 Most of the bladder cancer cases occur in men. In the US, the number of these patients is estimated to be 83,730. 5,6 In the middle-aged and older adult men, bladder cancer is the second most common malignancy. 7 There are several risk factors for initiation and development of bladder cancer including smoking, age, family history, exposure to chemicals and dyes, and chronic bladder inflammation. 8 The prognosis of patients with NMIBC is desirable and by local treatment, the transformation of NMIBC to MIBC is prevented and on time therapy inhibits tumor recurrence. Due to the development of metastasis, the prognosis of patients with MIBC is unfavorable and in addition to resection, patients receive adjuvant chemotherapy. 9 However, treatment appears not to be completely effective and patients present development of metastasis in spite of aggressive therapy. 10 In addition to surgery and chemotherapy, radiotherapy and immunotherapy in certain cases can be employed for the treatment of bladder cancer. 11,12 There is also a classification of bladder cancer based on the manner of tumor development including squamous cell carcinoma and carcinoma in mucus-secreting cells of the bladder which indeed is rare. 13 Despite various therapeutic strategies for bladder cancer, tumor cells have capacity of developing resistance to therapy. 14,15 Therefore, novel modalities for treatment of bladder cancer should be followed. Furthermore, different diagnostic tools including magnetic resonance imaging (MRI), computed tomography (CT) and chest X-ray, among others are utilized for bladder cancer detection. However, they also have their drawbacks such as low specificity and heterogeneous nature of bladder tumor cells. 16 Radical cystectomy is the gold standard for bladder cancer treatment. Despite the malignancy of tumor cells, the 5-year survival rate of patients with bladder cancer is estimated to be 20%-40%. 17,18 In order to improve the potential of therapy for bladder cancer, combination therapies with methotrexate, vinblastine, doxorubicin, and cisplatin are recommended. 19 However, in high-risk patients that have undergone radical cystectomy, the combination of cisplatin and gemcitabine for treatment of bladder cancer and improving overall survival is not significant. 19 Based on estimates, it has been reported that only 50% of bladder cancer patients respond to cisplatin-mediated chemotherapy. 20 Compared to other regimens, the combination of cisplatin and gemcitabine demonstrates low side effects and a high potential in the treatment of bladder cancer and retardation of metastasis. 21,22 In addition to chemotherapy and radical cystectomy, pelvic lymph node dissection is also performed for treatment of bladder cancer. 23,24 For patients that undergo surgery, radiotherapy is also used as a treatment modality, and in combination with chemotherapy, effectiveness of the therapy increases. 25 Despite significant efforts in the application of radiotherapy and chemotherapy as well as surgery in bladder cancer treatment, there are still challenges due to adverse impacts, resistance development, and metastatic nature of tumor cells. Metastasis of bladder tumor cells limits the application of surgery in bladder cancer treatment. Both radiotherapy and chemotherapy exhibit pronounced side effects in major organs. The potential of radiotherapy and chemotherapy in bladder cancer suppression is restricted by resistance development. 21,26,27 One of the new emerging therapeutic approaches in bladder cancer is the application of immunotherapy. In recent years, various kinds of checkpoint inhibitors including PD-L1/PD1, CTLA-4 as well as CAR T cell therapy have been developed for bladder cancer. However, immune evasion has reduced potential of immunotherapy in bladder cancer. 28 Nanotechnology is an interdisciplinary field that has opened the way for treatment and diagnosis of various diseases such as infectious diseases and cancer. [29][30][31][32][33][34][35][36][37][38][39] Nanostructures possess sizes at nm ranges and therefore, they can be easily internalized by tumor cells. Nanostructures can be employed for the purpose of cargo (drug and gene) delivery, immunotherapy, phototherapy, and diagnosis. The purpose of current review is to provide a comprehensive discussion of applications of nanomaterials in bladder cancer therapy and diagnosis.

| NANOPLATFORMS IN CHEMOTHERAPEUTIC DRUG DELIVERY
One of the optimal treatment approaches in cancer therapy is the application of chemotherapeutic agents. Intravenous injection is the main approach for the administration of these anti-cancer drugs.
However, systemic toxicity is mainly followed upon the application of chemotherapeutic agents, and it is recommended to use a low amount of these agents. Regardless of the adverse impacts of chemotherapeutic agents, frequent applications of these agents lead to the emergence of drug resistance. Therefore, solutions to these issues should be provided. In the first step, low concentration of chemotherapeutic agents should be employed and in the second step, combination therapy and increased accumulation of drugs at tumor site should be followed to prevent drug resistance. Emerging nanostructures have provided a great opportunity for delivery of chemotherapeutic agents to reduce their concentration (low level is loaded on nanocarriers), provide their targeted delivery, enhance their internalization in cancer cells, and prevent development of drug resistance. [40][41][42] This section focuses on nano-scale delivery systems for delivery of chemotherapeutic agents in bladder cancer therapy.
Among various kinds of chemotherapeutic agents employed for bladder cancer treatment, doxorubicin (DOX) is the most well-known medication capable of suppressing tumor progression via binding to topoisomerase enzymes to counter DNA replication and cancer cell proliferation. [43][44][45] Mesoporous silica nanoparticles (MSNs), modified with polydopamine and peptide, have been used for delivery of DOX.
Polydopamine modification of MSNs is of importance for providing sustained release of DOX and its pH-sensitive release, while peptide provides specific targeting of bladder tumor cells by binding to receptor. The drug-loaded MSNs had a particle size of 170.2 nm, zeta potential of À15.9 mV, and drug loading of 16.25%. These nanoparticles demonstrated high internalization capacity in bladder tumor cells, and they improved potency of DOX in bladder tumor suppression in vivo. 46 An interesting point is the increased anticancer activity of DOX against bladder tumors and reduced adverse impacts following encapsulation of DOX using nanostructures. 47 Lipid-based nanostructures are also promising candidates for DOX delivery in bladder cancer. Cationic micelles were prepared from 1,2-dioleoyl-3-trimethylammonium propane/methoxypoly (ethyleneglycol) (DPP) for DOX delivery and resulting nanocarriers were 18.65 nm in size and +19.6 mV in zeta potential. The cellular uptake and accumulation of DOX in bladder tumor cells were significantly enhanced, and these nanoparticles promoted residence of DOX in bladder. The in vivo experiment revealed boosted anticancer activity of DOX against bladder tumor. 48 In addition to micelles, other kinds of lipid-based nanostructures, known as liposomes, can be employed in DOX delivery. At the first step, folatemodified thermosensitive liposomes were fabricated and then, DOX, gold nanorods, and magnetic nanoparticles were loaded into these liposomes. This nanocomplex showed a particle size of 230 nm with superparamagnetic features capable of loading 0.57 mg/ml of DOX. The photothermal impact and alterations in temperature affect the release of DOX from these liposomal nanoparticles. After irradiation, 95% of DOX was released in 3 h, and due to modification with folic acid, the nanostructures bound to folate receptors on the surface of bladder cancer cells, resulting in an increase in their internalization. 49 Nanodiamonds (NDs) are a kind of solid nanostructures deemed ideal candidates for drug delivery and biomedical application courtesy of their high surface area and high drug loading efficiency. [50][51][52] The availability of NDs has opened a new window in cancer treatment due to their capacity in reversing chemoresistance. 52,53 However, the stability of NDs is a troublesome problem limiting their potential in cargo delivery. 54,55 It has been reported that coating NDs with polymers is a promising strategy to improve their stability. Chitosan-modified NDs were synthesized for intravesical delivery of DOX in bladder cancer therapy. These nanostructures demonstrated high drug loading efficiency (more than 90%) with an average particle size of 150 nm. The chitosan-modified NDs present great colloidal stability and desirable drug release, but their stability is relatively low in cultured media.
These DOX-loaded chitosan-modified NDs significantly suppressed bladder tumor progression ex vivo and provided high drug retention. 56 In fact, the ultimate goal of nanoscale delivery for DOX is to improve and to prolong its retention in bladder tumor site to decrease tumor cell viability up to 99%. 57 Another option in bladder cancer chemotherapy is cisplatin (CP) which is commonly used for locally advanced or metastatic tumors. 22 However, clinical trials show limitations of using CP for purpose of bladder cancer chemotherapy and to overcome the drawbacks, intravesical delivery of CP is suggested. CP-loaded nanostructures have shown potential for treatment of bladder cancer in clinical trials and reducing the adverse impacts of CP. [58][59][60][61][62][63][64][65] Coating nanomaterials with PEG enhances local delivery of CP and improves survival of animal models. 66 In an effort, CP was conjugated to poly(L-aspartic acid sodium salt) (PAA) or methoxy-poly(ethyleneglycol)-block-PAA (PEG-PAA) polymers with low or high PEG content. The intravesical administration of these nanoparticles significantly diminished side effects of CP and improved its safety profile. It is worth mentioning that administration of CP is correlated with hyperplasia and overweight of bladder tissue, while CP-loaded nanocarriers demonstrated high biocompatibility and lack of such drawbacks. CP-loaded nanocarriers had a particle size of 140 nm, zeta potential of À3.3 mV with drug loading up to 40%. These nanoparticles demonstrated great anti-proliferative activity against bladder cancer. Notably, CP-loaded PAA-modified nanostructures increased CP concentration in bladder tissue, while PEG-modified nanoparticles had no effect. 67 Polymeric nanoparticles and their combination with superparamagnetic iron oxide nanostructures (SPIONs) can be employed for CP delivery in bladder cancer therapy to offer an adjusted release.
First, PCL-b-P(PMA-click-MSA-co-PEGMA) nanoparticles were synthesized using three main strategies including ring-opening polymerization, reversible addition-fragmentation chain transfer (RAFT) polymerization, and thiol-yne "click" reaction. At the next step, SPIONs were embedded into PCL core, whereas CP was conjugated to surface of nanostructures via binding to dicarboxylic groups. These nanoparticles with a particle size and zeta potential of 281 nm and À34.3 mV, respectively, demonstrated mucoadhesive and superparamagnetic characteristics. After the administration of CP-loaded polymeric nanoparticles, 30% drug release (CP) occurred during first 4 h and then, prolonged release of CP was maintained for 4 days. The increase in temperature promoted the release of CP from nanostructures and they showed high anti-cancer activity against bladder tumor. 68 In addition to drug delivery, several nanoparticles such as cuprous oxide nanostructures have anti-tumor activity and increase ROS generation. These nanoparticles evoke apoptosis in reducing progression of bladder tumor cells, providing a synergy with chemotherapeutic agents such as CP and gemcitabine. 69 Therefore, targeted delivery of anti-cancer agents by nanostructures significantly promotes their anti-proliferative activity in bladder cancer. [70][71][72] Interestingly, nanocarriers can provide a platform for co-delivery of chemotherapeutic agents. Several experiments have revealed that co-delivery of synthetic drugs with nanostructures provides synergistic therapy in bladder cancer. Chitosan-polymethacrylic acid (CM) nanocapsules with capacity of attaching to luminal surface of bladder tissue and lack of damage to urothelium showed high biocompatibility and safety profile. These nanocapsules were prepared via electrostatic interaction between chitosan and methacrylic acid (MAA) chains. Then, CP and DOX as anti-cancer agents were loaded on these nanoparticles. This combination demonstrated 5-to 16-fold increase in anti-cancer activity compared to CP or DOX alone, and they could be internalized in bladder tumor cells with a high efficiency. DOX-and CP-loaded nanocapsules had a zeta potential of +15 mV, confirming their high stability. 73 Usually, nanoparticles employed for co-delivery of synthetic drugs have a high encapsulating efficiency (70% or more), and the only limitation is the increase in particle size due to co-loading, necessitating the need for the understanding of how enhancement in particle size can affect internalization of nanocarriers in bladder cancer cells. 74,75 The previous discussions revealed the role of nano-scale delivery systems for synthetic drugs in increasing their anti-cancer activity.
Noteworthy, nanoparticles can also increase bioavailability and therapeutic index of phytochemicals in bladder cancer therapy. The natural products have benefits such as low cost, multi-targeting capacity, and high safety profile that have made them appropriate options in cancer therapy. 76,77 However, accumulation of natural products in bladder tumor cells is low, urging scientists to find novel methods for their delivery. PLA-based nanostructures were prepared using precipitation technique and loaded with glycoalkaloidic extract (AE). The nanoparticles displayed a particle size of 200 nm, zeta potential of À12 and À7 mV, and encapsulation efficiency of 85%-90%. These AE-loaded nanostructures suppressed bladder cancer progression in a concentration-dependent manner and stimulated both apoptosis and cell cycle arrest. 78 In another experiment, nanoemulsions were synthesized from nonionic surfactant and Neem seed oil, and then, resveratrol as a phytochemical, was loaded in these nanostructures. The nanoparticles had a particle size of 137.8 nm, and they were stable for at least 30 days. They increased accumulation of resveratrol in bladder cancer cells and significantly reduced survival and viability of tumor cells. 79 The studies highlight the fact that delivery of phytochemicals with nanostructures is of importance in increasing their capacity for cell death induction and this is favorable for bladder cancer therapy ( Figure 1). 80

| NANOPLATFORMS IN GENE DELIVERY
In recent years, much attention has been directed toward the application of gene therapy for diseases, particularly cancer. 83 Genes have more specificity compared to drugs in targeting a specific molecular pathway and therefore, they are of high importance in precision medicine. 43 To date, various kinds of gene therapy approaches have been utilized for cancer treatment. The first kind of gene modality is the application of RNA interference (RNAi) for reducing expression level of genes. The siRNA and shRNA are two tools employed in treatment of cancer and by silencing target gene, depending on the function of genes, these tools significantly suppress proliferation and invasion of cancers. Noteworthy, siRNA and shRNA are beneficial in reversing drug resistance in tumor cells. 44,[84][85][86][87] Therefore, their application in cancer therapy can pave the way for better prognosis for cancer patients. Another approach for gene therapy is targeting noncoding RNAs (ncRNAs). These RNA molecules do not translate into proteins and they are involved in evolutionary mechanisms and regulation of biological events such as proliferation, migration, and differentiation. [88][89][90] The microRNAs (miRs) are the most wellknown members of ncRNAs and they participate in pathological events. Aberrant expression of miRNAs is associated with cancer F I G U R E 1 Nanomaterials for the delivery of chemotherapeutic agents in bladder cancer. The cationic micelles, mesoporous silica nanoparticles, liposomes, and chitosan nanoparticles among others, can be employed for the delivery of drugs such as cisplatin in cancer therapy to induce DNA damage and apoptosis and suppress proliferation of tumor cells. DOX, doxorubicin; PAA, poly(L-aspartic acid sodium salt); PEG, polyethylene glycol development and significant effort has been made in regulating their expression level. 91 Although gene therapy has opened a new gate in tumor therapy, this method has faced its own problems that should be overcome. The primary obstacle in gene therapy is the low accumulation of nucleic acid drugs within tumor tissues. For instance, blood-brain barrier (BBB) prevents the entrance of agents into brain and genetic tools have poor efficacy in the treatment of brain tumors. Furthermore, the presence of blood-tumor barrier (BTB) serves as an impediment to internalization of nucleic acid drugs to tumor cells. In vitro studies demonstrate a high efficacy of genetic tools although the application of these genes for in vivo results in their degradation in blood circulation by RNase enzymes. Therefore, encapsulation of nucleic acid drugs not only promotes their intracellular accumulation at tumor tissue, but also protects them against degradation. 44,45,[85][86][87] Therefore, current section focuses on the application of nanomaterials for genes delivery in bladder cancer therapy.
Chitosan-hyaluronic acid dialdehyde (HAD) nanostructures can deliver Bcl-2-siRNA to bladder cancer site and enable the suppression of tumor growth. HAD was prepared using ethanol-water mixture and it was conjugated to chitosan nanostructures. Then, these nanoparticles provided selective delivery of siRNA to bladder cancer cells overexpressing CD44. siRNA-loaded nanoparticles demonstrated particle size of 100-120 nm with a loading efficiency of 95% for siRNA. They had high biocompatibility and stability, and by binding to Exosomes are emerging nanostructures belonging to extracellular vesicles (EVs) with sizes smaller than 100 nm and can transfer lipids, proteins, and nucleic acids among cells. 95 Exosomes are potential therapeutic agents in bladder cancer, and they can be used for siRNA delivery in tumor suppression. As exosomes are derived from sources in body such as mesenchymal stem cells (MSCs), they have high biocompatibility and their safety profile for clinical trials has been confirmed. Exosomes derived from human embryonic kidney cells and MSCs were loaded with PLK-1 siRNA, and compared to normal epithelial cells, high amount of exosomes was internalized in bladder tumor cells. Through increased delivery and accumulation of siRNA in tumor cells, they provided effective gene silencing and reduced progression of bladder cancer cells. 96 Regardless of engineering exosomes for purpose of gene delivery, they can be secreted by cells in body, and by transferring, for instance, miRNA-4792, they can enhance progression of bladder tumor cells via inducing c-Myc signaling. 97 In this condition, preventing biogenesis and secretion of exosomes is preferred. However, the purpose of this review is on engineered exosomes as nanostructures in gene delivery for bladder cancer therapy.
Based on these findings, application of siRNA is of importance for the suppression of proliferation and metastasis of bladder cancer cells, and their efficiency in gene silencing enhances using nanoparticles for targeted delivery. 98,99 As it was mentioned in the introduction section, chemoresistance is an increasing challenge in bladder cancer, and overcoming drug resistance requires identification of factors involved in this process and their targeting. 100 Nrf2 is an oxidative stress regulator and is involved in reducing ROS levels via reinforcing antioxidant defense system. The overexpression of Nrf2 protein is beneficial for protection of normal cells; however, its upregulation induces resistance of tumor cells to cytotoxic impact of anti-cancer agents. 45 Therefore, downregulation of Nrf2 is an ideal strategy in cancer therapy. For this purpose, guanidine-terminated carbosilane dendrimers   (Table 1).

| NANOPLATFORMS IN PHOTOTHERAPY
The red-light irradiation was first used in 1975 in killing tumor cells.
Since then, photodynamic therapy (PDT) has been considered as a promising strategy in the treatment of cancer. 121 Three main components are required for an appropriate PDT including photoactive drug, light with a proper wavelength, and molecular oxygen. 121 Upon exposure to light, excitation of photoactive drug to its triplet state occurs that interacts with oxygen in tissue, leading to generation of reactive oxygen species (ROS). PDT is considered as one of the most promising approaches in treatment of cancer and it has low side effects and partially affects neighboring cells and tissues. 122 Photothermal therapy (PTT) is also another approach for minimally-invasive treatment of cancer that uses a photothermal agent for converting light to energy to eliminate tumor cells. 123  co-utilized to suppress progression and viability of tumor cells in a synergistic manner. [124][125][126][127] The current section focuses on the application of PDT and PTT in the treatment of bladder cancer.
Currently, surgery is not considered as a promising option in bladder cancer treatment due to tumor recurrence and metastasis, and its combination with chemotherapy has other problems such as side effects. Therefore, innovative approaches such as PTT are employed Single-walled carbon nanotubes (SWCNTs) have been employed for DOX delivery and providing PTT. The drug loading efficiency of SWCNTs was 40% and they had a particle size of 220 nm. These nanostructures significantly accumulated at tumor site, and by providing chemotherapy and PTT, they suppressed the progression of bladder cancer. 137 Hence, combination of chemotherapy and phototherapy shows some promising results in bladder cancer therapy. 138 This benefit has been confirmed in both in vitro and in vivo studies. 139 Another issue worth mentioning for the application of phototherapy is its impact on immune system. It has been reported that PDT has the capacity of ROS overgeneration and enhancing anti-tumor immunity via T cell infiltration. However, PDT can enhance PD-L1 expression to mediate immunosuppression. Liposomes were used to codeliver metformin and IR-775 to enhance ROS overgeneration and down-regulate PD-L1 expression to mediate PDT and immunotherapy in bladder cancer ( Figure 5). 140 Therefore, another approach is combination of phototherapy and immunotherapy in suppressing bladder tumor progression (

| STIMULI-RESPONSIVE NANOPLATFORMS
In recent years, the application of smart and advanced nanoplatforms in cancer therapy has stirred much interest. 159 The current section shows the pathway followed by nanocarriers for improving the internalization of therapeutic modalities in bladder cancer.
There are specific receptors on the surface of cells that are responsible for uptake of nanostructures through a mechanism, known as endocytosis. 184 Nanomaterials have size and dimensions equivalent to intracellular organelles, and they may affect biological processes. Due to the interaction between living organisms and nanoparticles, cellular physiology is greatly affected and this interaction can be positive or negative. 185 Both modified and nonmodified cles. 186,187 The pinocytosis and phagocytosis are two major kinds of endocytic pathway, and their difference is in the size of nanoparticles that they internalize. The particles with small size are internalized with pinocytosis, whereas large particles with sizes more than 500 nm are internalized via phagocytosis. Besides, pinocytosis is divided into clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-and caveolae-independent endocytosis. 188 The clathrinmediated endocytosis and caveolae-mediated endocytosis pathways are energy-dependent and are responsible cellular uptake of biomolecules. The macropinocytosis is independent of clathrin-mediated and caveolae-mediated endocytosis, and they are driven by actin filaments ( Figure 10). 189,190 The shape, size, and surface charge of nanoparticles are among   Table 3

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.